Method of use of peptide antagonists of zonulin to prevent or delay the onset of diabetes

ABSTRACT

A method for prevention of delaying the onset of diabetes using peptide antagonists of zonulin, is disclosed.

The development of the present invention was supported by the Universityof Maryland, Baltimore, Md. The invention described herein was supportedby funding from the National Institutes of Health (DK 48373-05). TheGovernment has certain rights.

FIELD OF THE INVENTION

The present invention relates to use of peptide antagonists of zonulinto prevent or delay the onset of diabetes, particularly type I diabetes.The peptide antagonists bind to the zonula occludens toxin receptor, yetdo not physiologically modulate the opening of mammalian tightjunctions.

BACKGROUND OF THE INVENTION

I. Function and Regulation of Intestinal Tight Junctions

The intestinal epithelium represents the largest interface (more than2,000,000 cm²) between the external environment and the internal milieu.The maintenance of intercellular tight junctions (“tj”) competenceprevents movements of potentially harmful environmental factors, such asbacteria, viruses, toxins, food allergens, and macromolecules across theintestinal barrier. This competence is significantly jeopardized in avariety of clinical conditions affecting the gastrointestinal tract,including food allergies, enteric infections, malabsorption syndromes,and inflammatory bowel diseases.

The tj or zonula occludens (hereinafter “ZO”) are one of the hallmarksof absorptive and secretory epithelia (Madara, J. Clin. Invest.,83:1089-1094 (1989); and Madara, Textbook of Secretory Diarrhea Eds.Lebenthal et al, Chapter 11, pages 125-138 (1990)). As a barrier betweenapical and basolateral compartments, they selectively regulate thepassive diffusion of ions and water-soluble solutes through theparacellular pathway (Gumbiner, Am. J. Physiol., 253 (Cell Physiol.22):C749-C758 (1987)). This barrier maintains any gradient generated bythe activity of pathways associated with the transcellular route(Diamond, Physiologist, 20:10-18 (1977)).

Variations in transepithelial conductance can usually be attributed tochanges in the permeability of the paracellular pathway, since theresistances of enterocyte plasma membranes are relatively high (Madara(1989, 1990), supra). The ZO represents the major barrier in thisparacellular pathway, and the electrical resistance of epithelialtissues seems to depend on the number of transmembrane protein strands,and their complexity in the ZO, as observed by freeze-fracture electronmicroscopy (Madara et al, J. Cell Biol., 101:2124-2133 (1985)).

There is abundant evidence that ZO, once regarded as static structures,are in fact dynamic and readily adapt to a variety of developmental(Magnuson et al, Dev. Biol., 67:214-224 (1978); Revel et al, Cold SpringHarbor Symp. Quant. Biol., 40:443-455 (1976); and Schneeberger et al, J.Cell Sci., 32:307-324 (1978)), physiological (Gilula et al, Dev. Biol.,50:142-168 (1976); Madara et al, J. Membr. Biol., 100:149-164 (1987);Mazariegos et al, J. Cell Biol., 98:1865-1877 (1984); and Sardet et al,J. Cell Biol., 80:96-117 (1979)), and pathological (Milks et al, J. CellBiol., 103:2729-2738 (1986); Nash et al, Lab. Invest., 59:531-537(1988); and Shasby et al, Am. J. Physiol., 255(Cell Physiol.,24:C781-C788 (1988)) circumstances. The regulatory mechanisms thatunderlie this adaptation are still not completely understood. However,it is clear that, in the presence of Ca²⁺, assembly of the ZO is theresult of cellular interactions that trigger a complex cascade is ofbiochemical events that ultimately lead to the formation and modulationof an organized network of ZO elements, the composition of which hasbeen only partially characterized (Diamond, Physiologist, 20:10-18(1977)). A candidate for the transmembrane protein strands, occluden,has recently been identified (Furuse et al, J. Membr. Biol., 87:141-150(1985)).

Six proteins have been identified in a cytoplasmic submembranous plaqueunderlying membrane contacts, but their function remains to beestablished (Diamond, supra). ZO-1 and ZO-2 exist as a heterodimer(Gumbiner et al, Proc. Natl. Acad. Sci., USA, 88:3460-3464 (1991)) in adetergent-stable complex with an uncharacterized 130 kD protein (ZO-3).Most immunoelectron microscopic studies have localized ZO-1 to preciselybeneath membrane contacts (Stevenson et al, Molec. Cell Biochem.,83:129-145 (1988)). Two other proteins, cingulin (Citi et al, Nature(London), 333:272-275 (1988)) and the 7H6 antigen (Zhong et al, J. CellBiol., 120:477-483 (1993)) are localized further from the membrane andhave not yet been cloned. Rab 13, a small GTP binding protein has alsorecently been localized to the junction region (Zahraoui et al, J. CellBiol., 124:101-115 (1994)). Other small GTP-binding proteins are knownto regulate the cortical cytoskeleton, i.e., rho regulatesactin-membrane attachment in focal contacts (Ridley et al, Cell,70:389-399 (1992)), and rac regulates growth factor-induced membraneruffling (Ridley et al, Cell, 70:401-410 (1992)). Based on the analogywith the known functions of plaque proteins in the better characterizedcell junctions, focal contacts (Guan et al, Nature, 358:690-692 (1992)),and adherens junctions (Tsukita et al, J. Cell Biol., 123:1049-1053(1993)), it has been hypothesize that tj-associated plaque proteins areinvolved in transducing signals in both directions across the cellmembrane, and in regulating links to the cortical actin cytoskeleton.

To meet the many diverse physiological and pathological challenges towhich epithelia are subjected, the ZO must be capable of rapid andcoordinated responses that require the presence of a complex regulatorysystem. The precise characterization of the mechanisms involved in theassembly and regulation of the ZO is an area of current activeinvestigation.

There is now a body of evidence that tj structural and functionallinkages exist between the actin cytoskeleton and the tj complex ofabsorptive cells (Gumbiner et al, supra; Madara et al, supra; andDrenchahn et al, J. Cell Biol., 107:1037-1048 (1988)). The actincytoskeleton is composed of a complicated meshwork of microfilamentswhose precise geometry is regulated by a large cadre of actin-bindingproteins. An example of how the state of phosphorylation of anactin-binding protein might regulate cytoskeletal linking to the cellplasma membrane is the myristoylated alanine-rich C kinase substrate(hereinafter “MARCKS”). MARCKS is a specific protein kinase C(hereinafter “PKC”) substrate that is associated with the cytoplasmicface of the plasma membrane (Aderem, Elsevier Sci. Pub. (UK), pages438-443 (1992)). In its non-phosphorylated form, MARCKS crosslinks tothe membrane actin. Thus, it is likely that the actin meshworkassociated with the membrane via MARCKS is relatively rigid (Hartwig etal, Nature, 356:618-622 (1992)). Activated PKC phosphorylates MARCKS,which is released from the membrane (Rosen et al, J. Exp. Med.,172:1211-1215 (1990); and Thelen et al, Nature, 351:320-322 (1991)). Theactin linked to MARCKS is likely to be spatially separated from themembrane and be more plastic. When MARCKS is dephosphorylated, itreturns to the membrane where it once again crosslinks actin (Hartwig etal, supra; and Thelen et al, supra). These data suggest that the F-actinnetwork may be rearranged by a PKC-dependent phosphorylation processthat involves actin-binding proteins (MARCKS being one of them).

A variety of intracellular mediators have been shown to alter tjfunction and/or structure. Tight junctions of amphibian gallbladder(Duffey et al, Nature, 204:451-452 (1981)), and both goldfish (Bakker etal, Am. J. Physiol., 246:G213-G217 (1984)) and flounder (Krasney et al,Fed. Proc., 42:1100 (1983)) intestine, display enhanced resistance topassive ion flow as intracellular cAMP is elevated. Also, exposure ofamphibian gallbladder to Ca²⁺ ionophore appears to enhance tjresistance, and induce alterations in tj structure (Palant et al, Am. J.Physiol., 245:C203-C212 (1983)). Further, activation of PKC by phorbolesters increases paracellular permeability both in kidney (Ellis et al,C. Am. J. Physiol., 263 (Renal Fluid Electrolyte Physiol. 32): F293-F300(1992)), and intestinal (Stenson et al, C. Am. J. Physiol.,265(Gastrointest. Liver Physiol., 28):G955-G962 (1993)) epithelial celllines.

II. Zonula Occludens Toxin

Most Vibrio cholerae vaccine candidates constructed by deleting the ctxAgene encoding cholera toxin (CT) are able to elicit high antibodyresponses, but more than one-half of the vaccinees still develop milddiarrhea (Levine et al, Infect. Immun., 56(1):161-167 (1988)). Given themagnitude of the diarrhea induced in the absence of CT, it washypothesized that V. cholerae produce other enterotoxigenic factors,which are still present in strains deleted of the ctxa sequence (Levineet al, supra). As a result, a second toxin, zonula occludens toxin(hereinafter “ZOT”) elaborated by V. cholerae and which contribute tothe residual diarrhea, was discovered (Fasano et al, Proc. Natl. Acad.Sci., USA, 8:5242-5246 (1991)). The zot gene is located immediatelyadjacent to the ctx genes. The high percent concurrence of the zot genewith the ctx genes among V. cholerae strains (Johnson et al, J. Clin.Microb., 31/3:732-733 (1993); and Karasawa et al, FEBS MicrobiologyLetters, 106:143-146 (1993)) suggests a possible synergistic role of ZOTin the causation of acute dehydrating diarrhea typical of cholera.Recently, the zot gene has also been identified in other entericpathogens (Tschape, 2nd Asian-Pacific Symposium on Typhoid fever andother Salomellosis, 47(Abstr.) (1994)).

It has been previously found that, when tested on rabbit ileal mucosa,ZOT increases the intestinal permeability by modulating the structure ofintercellular tj (Fasano et al, supra). It has been found that as aconsequence of modification of the paracellular pathway, the intestinalmucosa becomes more permeable. It also was found that ZOT does notaffect Na⁺-glucose coupled active transport, is not cytotoxic, and failsto completely abolish the transepithelial resistance (Fasano et al,supra).

More recently, it has been found that ZOT is capable of reversiblyopening tj in the intestinal mucosa, and thus ZOT, when co-administeredwith a therapeutic agent, e.g., insulin, is able to effect intestinaldelivery of the therapeutic agent, when employed in an oral dosagecomposition for intestinal drug delivery, e.g., in the treatment ofdiabetes (WO 96/37196; U.S. Pat. No. 5,827,534; U.S. Pat. No. 5,665,389;and Fasano et al, J. Clin. Invest., 99:1158-1164 (1997); each of whichis incorporated by reference herein in their entirety). It has also beenfound that ZOT is capable of reversibly opening tj in the nasal mucosa,and thus ZOT, when co-administered with a therapeutic agent, is able toenhance nasal absorption of a therapeutic agent (U.S. Pat. No.5,908,825; which is incorporated by reference herein in its entirety).

In U.S. Pat. No. 5,864,014; which is incorporated by reference herein inits entirety, a ZOT receptor has been identified and purified from anintestinal cell line, i.e., CaCo2 cells. Further, in U.S. Pat. No.5,912,323; which is incorporated by reference herein in its entirety,ZOT receptors from human intestinal, heart and brain tissue have beenidentified and purified. The ZOT receptors represent the first step ofthe paracellular pathway involved in the regulation of intestinal andnasal permeability.

III. Zonulin

In U.S. Pat. Nos. 5,945,510 and 5,948,629, which are incorporated byreference herein in their entirety, mammalian proteins that areimmunologically and functionally related to ZOT, and that function asthe physiological modulator of mammalian tight junctions, have beenidentified and purified. These mammalian proteins, referred to as“zonulin”, are useful for enhancing absorption of therapeutic agentsacross tj of intestinal and nasal mucosa, as well as across tj of theblood brain barrier.

IV. Peptide Antagonists of Zonulin

Peptide antagonists of zonulin were identified and described for thefirst time in pending U.S. patent application Ser. No. 09/127,815, filedAug. 3, 1998, which is incorporated by reference herein in its entirety,which corresponds to WO 00/07609. Said peptide antagonists bind to theZOT receptor, yet do not function to physiologically modulate theopening of mammalian tight junctions. The peptide antagonistscompetitively inhibit the binding of ZOT and zonulin to the ZOTreceptor, thereby inhibiting the ability of ZOT and zonulin tophysiologically modulate the opening of mammalian tight junctions.

V. Diabetes

The morbidity and mortality associated with diabetes is devastating. Thetotal number of diabetic individuals in the United States is 15.7million. Of these, 100% of the type I diabetic individuals and 40% oftype II diabetic individuals depend on paternal administration ofinsulin. On an annual basis, the direct medical costs associated s withdiabetes exceeds 40 billion dollars. An additional 14 billion dollars isassociated with disability, work loss, and premature mortality.

Although oral insulin drug delivery strategies have been the focus ofmany research efforts, they have been largely unsuccessful because thephysiologic nature of the small intestine prevents the absorption ofmacromolecules, such as insulin.

An oral dosage composition comprising ZOT for targeting delivery ofinsulin to the paracellular pathway for the treatment of diabetes hasbeen described in U.S. Pat. Nos. 5,827,534 and 5,665,389. Byphysiologically modulating the paracellular pathway using ZOT, it is nowpossible to introduce a wide variety of therapeutic agents into thesystemic circulation. This drug delivery system adds targetingspecificity, which has long hampered the design of many oralpharmaceutical agents. The utility of this system is not limited toinsulin delivery, and may represent a new way of designing orallyadministered pharmaceutical agents.

While offering an innovative treatment strategy for a disease asdebilitating as diabetes is promising, preventing or delaying the onsetof disease has widespread implications. Understanding the pathogenesisof any disease process is a daunting task. Heretofore, there has been noprior evidence of a pharmaceutical agent with the capability ofpreventing or delaying the onset of diabetes. In the present inventionnew light has been shed on the pathogenesis, prevention and delaying ofonset of diabetes by demonstrating that a critical and early step indisease progression resides in alterations in paracellular permeability.In the present invention, it has been demonstrated that an increase inparacellular permeability is necessary for the progression towarddiabetes. Peptide antagonists of zonulin, which block this endogenouspathway, have been found in the present invention to prevent theprogression to diabetes. Thus, the present invention is believed to beuseful to prevent long-term complications of diabetes. Further, thepermeability changes associated with autoimmune diseases are longstanding, and early intervention per the present invention is believedto have untold benefits to the diabetic patient.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for theprevention or delay the onset of diabetes.

This and other objects of the present invention, which will be apparentfrom the detailed description of the invention provided hereinafter,have been met, in one embodiment, by a method for preventing or delaythe onset of diabetes (particularly, type I diabetes) comprisingadministering to a subject in need of such prevention or delay of onset,a pharmaceutically effective amount of a peptide antagonist of zonulin,wherein said peptide antagonist binds to ZOT receptor, yet does notphysiologically modulate the opening of mammalian tight junctions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the N-terminal sequences of zonulinpurified from various human tissues and IgM heavy chain with theN-terminal sequence of the biologically active fragment (amino acids288-399) of ZOT.

FIG. 2 shows the effect of ZOT, zonulin_(i), zonulin_(h), either alone(closed bars), or in combination with the peptide antagonist FZI/0 (openbars) or in combination with FZI/1 (shaded bars), as compared to thenegative control, on the tissue resistance (Rt) of rabbit ileum mountedin Ussing chambers. N equals 3-5; and * equals p<0.01.

FIG. 3 shows the concentrations (ng/ml) of intraluminal zonulin in bothdiabetic-prone and diabetic-resistant rats, which was determined using asandwich ELISA assay. Samples were obtained by intestinal lavage innormal saline. The first bar in each case represents diabetic-resistantrats (DR). The second bar represents diabetic-prone animals (DP), andthe third bar represents rats with chronic diabetes (CD). <9% of thediabetic-prone rats do not become diabetic, and <9% of thediabetic-resistant rats develop diabetes.

FIG. 4 shows the percentage of rats used in the study that progressed todiabetes.

FIG. 5 shows the concentrations (ng/ml) of intraluminal zonulin indiabetic rats, which was determined using a sandwich ELISA assay.

FIG. 6 shows ex vivo intestinal permeability in diabetic resistant (DR)rats, untreated diabetic-prone rats (DP-untreated; second bar)determined in Ussing chambers, diabetic-prone rats treated with thepeptide antagonist of zonulin (DP-treated; third bar). * equals p<0.05;** equals p<0.05, and p<0.0001 compared to DP-treated.

FIG. 7 shows ex vivo intestinal permeability in the small intestines ofuntreated diabetes-prone rats that either developed or did not developdiabetes. * equals p<0.04.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, in one embodiment, the above-described object of thepresent invention have been met by a method for preventing or delayingthe onset of diabetes (particularly, type I diabetes) comprisingadministering to a subject in need of such prevention or delay of onset,a pharmaceutically effective amount of a peptide antagonist of zonulin,wherein said peptide antagonist binds to ZOT receptor, yet does notphysiologically modulate the opening of mammalian tight junctions.

The particular peptide antagonist of zonulin employed in the presentinvention is not critical thereto. Examples of said peptide antagonistsinclude peptides which comprise an amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.

The size of the peptide antagonist is not critical to the presentinvention. Generally, the size of the peptide antagonist will range from8 to 110, amino acids, preferably from 8 to 40 amino acids, morepreferably will be 8 amino acids.

The peptide antagonists can be chemically synthesized and purified usingwell-known techniques, such as described in High Performance LiquidChromatography of Peptides and Proteins: Separation Analysis andConformation, Eds. Mant et al, C.R.C. Press (1991), and a peptidesynthesizer, such as Symphony (Protein Technologies, Inc); or by usingrecombinant DNA techniques, i.e., where the nucleotide sequence encodingthe peptide is inserted in an appropriate expression vector, e.g., an E.coli or yeast expression vector, expressed in the respective host cell,and purified therefrom using well-known techniques.

The peptide antagonists can be administered as oral dosage compositionsfor small intestinal delivery. Such oral dosage compositions for smallintestinal delivery are well-known in the art, and generally comprisegastroresistent tablets or capsules (Remington's PharmaceuticalSciences, 16th Ed., Eds. Osol, Mack Publishing Co., Chapter 89 (1980);Digenis et al, J. Pharm. Sci., 83:915-921 (1994); Vantini et al, ClinicaTerapeutica, 145:445-451 (1993); Yoshitomi et al, Chem. Pharm. Bull.,40:1902-1905 (1992); Thoma et al, Pharmazie, 46:331-336 (1991);Morishita et al, Drug Design and Delivery, 7:309-319 (1991); and Lin etal, Pharmaceutical Res., 8:919-924 (1991)); each of which isincorporated by reference herein in its entirety).

Tablets are made gastroresistent by the addition of, e.g., eithercellulose acetate phthalate or cellulose acetate terephthalate.

Capsules are solid dosage forms in which the peptide antagonist(s) isenclosed in either a hard or soft, soluble container or shell ofgelatin. The gelatin used in the manufacture of capsules is obtainedfrom collagenous material by hydrolysis. There are two types of gelatin.Type A, derived from pork skins by acid processing, and Type B, obtainedfrom bones and animal skins by alkaline processing. The use of hardgelatin capsules permit a choice in prescribing a single peptideantagonist or a combination thereof at the exact dosage level consideredbest for the individual subject. The hard gelatin capsule consists oftwo sections, one slipping over the other, thus completely surroundingthe peptide antagonist. These capsules are filled by introducing thepeptide antagonist, or gastroresistent beads containing the peptideantagonist, into the longer end of the capsule, and then slipping on thecap. Hard gelatin capsules are made largely from gelatin, FD&Ccolorants, and sometimes an opacifying agent, such as titanium dioxide.The USP permits the gelatin for this purpose to contain 0.15% (w/v)sulfur dioxide to prevent decomposition during manufacture.

In the context of the present invention, oral dosage compositions forsmall intestinal delivery also include liquid compositions which containaqueous buffering agents that prevent the peptide antagonist from beingsignificantly inactivated by gastric fluids in the stomach, therebyallowing the peptide antagonist to reach the small intestines in anactive form. Examples of such aqueous buffering agents which can beemployed in the present invention include bicarbonate buffer (pH 5.5 to8.7, preferably about pH 7.4).

When the oral dosage composition is a liquid composition, it ispreferable that the composition be prepared just prior to administrationso as to minimize stability problems. In this case, the liquidcomposition can be prepared by dissolving lyophilized peptide antagonistin the aqueous buffering agent.

The pharmaceutically effective amount of peptide antagonist employed isnot critical to the present invention and will vary depending upon theage, weight and sex of the subject being treated. Generally, the amountof peptide antagonist employed in the present invention to prevent ordelay the onset of diabetes, is in the range of about 7.5×10⁻⁶ M to7.5×10⁻³ M, preferably about 7.5×10⁻⁶ M to 7.5×10⁻⁴ M. To achieve such afinal concentration in, e.g., the intestines or blood, the amount ofpeptide antagonist in a single oral dosage composition of the presentinvention will generally be about 1.0 μg to 1000 μg, preferably about1.0 μg to 100 μg.

The following examples are provided for illustrative purposes only, andare in no way intended to limit the scope of the present invention.

EXAMPLE 1 Peptide Antagonists of Zonulin

Given that ZOT, human intestinal zonulin (zonulin_(i)) and human heartzonulin (zonulin_(h)) all act on intestinal (Fasano et al,Gastroenterology, 112:839 (1997); Fasano et al, J. Clin. Invest., 96:710(1995)) and endothelial tj and that all three have a similar regionaleffect (Fasano et al (1997), supra) that coincides with the ZOT receptordistribution within the intestine (Fasano et al (1997), supra; andFasano et al (1995), supra), it was postulated in U.S. patentapplication Ser. No. 09/127,815, filed Aug. 3, 1998, that these threemolecules interact with the same receptor binding site. A comparison ofthe primary amino acid structure of ZOT and the human zonulins was thuscarried out therein to provide insights as to the absolute structuralrequirements of the receptor-ligand interaction involved in theregulation of intestinal tj. The analysis of the N-termini of thesemolecules revealed the following common motif (amino acid residues 8-15boxed in FIG. 1): non-polar (Gly for intestine, Val for brain),variable, non-polar, variable, non-polar, polar, variable, polar (Gly).Gly in position 8, Val in position 12 and Gln in position 13, all arehighly conserved in ZOT, zonulin_(i) and zonulin_(h) (see FIG. 1), whichis believed to be critical for receptor binding function within theintestine. To verify the same, the synthetic octapeptide Gly Gly Val LeuVal Gln Pro Gly (SEQ ID NO:15) (named FZI/0, and corresponding to aminoacid residues 8-15 of human fetal zonulin_(i)) was chemicallysynthesized.

Next, rabbit ileum mounted in Ussing chambers as described above, wereexposed to 100 μg of FZI/0 (SEQ ID NO:15), 100 μg of FZI/1 (SEQ IDNO:29), 1.0 μg of 6×His-ZOT (obtained as described in Example 1 of U.S.patent application Ser. No. 09/127,815, filed Aug. 3, 1998), 1.0 μg ofzonulin_(i) (obtained as described in Example 3 of U.S. patentapplication Ser. No. 09/127,815, filed Aug. 3, 1998), or 1.0 μg ofzonulin_(h) (obtained as described in Example 3 of U.S. patentapplication Ser. No. 09/127,815, filed Aug. 3, 1998), alone; orpre-exposed for 20 min to 100 μg of FZI/0 or FZI/1, at which time 1.0 μgof 6×His-ZOT, 1.0 μg of zonulin_(i), or 1.0 μg of zonulin_(h), wasadded. ΔRt was then calculated as described above. The results are shownin FIG. 2.

As shown in FIG. 2, FZI/0 did not induce any significant change in Rt(0.5% as compared to the negative control) (see closed bar). On thecontrary, pre-treatment for 20 min with FZI/0 decreased the effect ofZOT, zonulin_(i), and zonulin_(h) on Rt by 75%, 97%, and 100%,respectively (see open bar). Also as shown in FIG. 2, this inhibitoryeffect was completely ablated when a second synthetic peptide (FZI/1,SEQ ID NO:29) was chemically synthesized by changing the Gly in position8, the Val in position 12, and the Gln in position 13 (as referred tozonulin_(i)) with the correspondent amino acid residues of zonulin_(b)(Val, Gly, and Arg, respectively, see SEQ ID NO:30) was used (see shadedbar).

The above results demonstrate that there is a region spanning betweenresidue 8 and 15 of the N-terminal end of ZOT and the zonulin familythat is crucial for the binding to the target receptor, and that theamino acid residues in position 8, 12 and 13 determine the tissuespecificity of this binding.

EXAMPLE 2 Diabetic Rat Model

Alterations in intestinal permeability have been shown to be one of thepreceding physiologic changes associated with the onset of diabetes(Meddings, Am. J. Physiol., 276:G951-957 (1999)). Paracellular transportand intestinal permeability is regulated by intracellular tj viamechanisms which have not been completely elucidated.

Zonulin and its prokaryotic analog, ZOT, both alter intestinalpermeability by modulating tj. In this example, it has been demonstratedfor the first time that zonulin-related impairment of tj is involved inthe pathogenesis of diabetes, and that diabetes can be prevented, or theonset delayed, by administration of a peptide antagonist of zonulin.

Initially, two genetic breeds, i.e., BB/Wor diabetic-prone (DP) anddiabetic-resistant (DR) rats (Haber et al, J. Clin. Invest., 95:832-837(1993)), were evaluated to determine whether they exhibited significantchanges in intraluminal secretion of zonulin and intestinalpermeability.

More specifically, age-matched DP and DR rats (20, 50, 75, and >100 daysof age) were sacrificed. After the rats were sacrificed, a 25 G needlewas placed within the lumen of the ileum, and intestinal lavage withRinger's solution was performed to determine the presence ofintraluminal zonulin. Zonulin concentration was evaluated using asandwich enzyme linked immunosorbent assay (ELISA) as follows:

Plastic microtiter plates (Costar, Cambridge, MA) were coated withpolyclonal rabbit anti-ZOT antibodies (obtained as described in Example2 of U.S. application Ser. No. 09/127,815 filed Aug. 3, 1998) (dilution1:100) overnight at 4° C., washed three times with PBS containing 0.05%(v/v) Tween 20, then blocked by incubation with 300 μl of PBS containing0.1% (v/v) Tween 20, for 15 min at room temperature. Next, purifiedhuman intestine zonulin (obtained as described in Example 3 of U.S.application Ser. No. 09/127,815 filed Aug. 3, 1998) was coated on theplates.

A standard curve was obtained by diluting zonulin in PBS containing0.05% (v/v) Tween 20 at different concentration: 0.78 ng/ml, 1.56 ng/ml,3.125 ng/ml, 6.25 ng/ml, 12.5 ng/ml, 25 ng/ml and 50 ng/ml.

100 μl of each standard concentration or 100 μl of intestinal lavagesample were pipetted into the wells, and incubated for 1 hr at roomtemperature, using a plate shaker. Unbound zonulin was washed-out usingPBS, and the wells were incubated with 100 μl of anti-ZOT antibodiesconjugated with alkaline phosphate for 1 hr at room temperature withshaking. Unbound conjugate was washed-out with PBS, and a color reactionwas developed by first adding 100 μl of Extra-Avidin (SIGMA, St. Louis,Mo.) diluted 1/20000 in 0.1 M Tris-HCl (pH 7.3), 1.0 mM MgCl₂, 1.0%(w/v) BSA for 15 min, and then incubating each well for 30 min at 37° C.with 100 μl of a solution containing 1.0 mg/ml ofp-nitrophenyl-phosphate substrate (SIGMA, St Louis, Mo.). Adsorbance wasread on an enzyme immunoassay reader at 405 nm.

In order to evaluate the intra- and inter-assay precision of theELISA-sandwich method, the coefficient variation (CV) was calculatedusing three replicates from two samples with different concentrations ofzonulin, on three consecutive days. The inter-assay test of theELISA-sandwich method produced CV values of 9.8%. The CV of theintra-assay test was 4.2% at day 1, 3.3% at day 2 and 2.9% at day 3.

Zonulin concentration was expressed as ng/mg protein detected in theintestinal lavages and normalized by exposed surface area (in mm²). Theresults are shown in FIG. 3.

As shown in FIG. 3, a 4-fold increase in intraluminal zonulin was firstobserved in diabetic-prone rats (age 50 days) (second bar). Thisincrease in intraluminal zonulin was found to correlate with an increasein intestinal permeability. The increase in intraluminal zonulin remainshigh in these diabetic-prone rats, and found to correlate with theprogression toward full-blown diabetes. Of note, the diabetic-prone rat(age >100 days) did not have an increase in intraluminal zonulin. Thisis remarkable, as this rat did not progress to diabetes. Blood glucosefor this rat was normal. Thus, zonulin is responsible for thepermeability changes associated with the pathogenesis of type Idiabetes. The increase in zonulin secretion is age-related, and proceedsthe onset of diabetes.

Next, in order to demonstrate that diabetes can be prevented byadministration of a peptide antagonist of zonulin, BB/Wor rats (ages21-26 days), were obtained from Biomedical Research Models, Inc.(Rutland, Mass.), and were randomized into two groups (n=5 per group),i.e., a treated group and a control group. Both groups were maintainedon a standard diet of rat chow (Harlan Teklab Diet #7012). All food andwater were previously autoclaved. Each day, daily water intake wasmeasured and 100 ml of fresh water was given. The treated group received10 μg/ml of the zonulin peptide antagonist (SEQ ID NO:15) supplementedin the drinking water. The rats were housed in hepa-filter cages.

Diabetes in the rats was diagnosed as follows: The rats were weighedtwice a week. Blood glucose was determined weekly using the OneTouch®glucose monitoring system (Johnson & Johnson). Each week, reagent stripsfor urinalysis were used to monitor glucose (Diastix®) and ketones(Ketositx®) (Bayer). Rats with a blood glucose >250 mg/dl were fastedovernight, and blood glucose levels >200 mg/dl were considered diabetic.These guidelines are in accordance with the data supplied by BiomedicalResearch Models, Inc. The results are shown in FIG. 4.

As shown in FIG. 4, 80% of the control rats (4/5) and 40% of the ratstreated with the peptide antagonist of zonulin (2/5) developed diabetesby age 80 days. Alterations in zonulin secretion paralleled the onset ofdiabetes.

Following clinical presentation of diabetes, the rats were sacrificed asfollows: the rats were anesthesized using ketamine anesthesia and amidline incision was made allowing access to the heart. An 18 G needlewas placed into the heart and death occurred by exsanguinations. Then,zonulin assays were conducted as described above. For those rats thatdid not present with diabetes, the endpoint of the study was age 80days. According to Biomedical Research Models, Inc., 80% of diabetesprone rats. present with diabetes by age 80 days. The results of thezonulin assays are shown in FIG. 5.

As shown in FIG. 5, the diabetic rats that were not treated with thepeptide antagonist of zonulin were observed to have an increase inintraluminal zonulin, which was consistent with the results shown inFIG. 3. Further, intraluminal zonulin was increased 2 to 4-fold indiabetic rats (DR), as compared to both diabetic-prone rats that did notdevelop diabetes (DP-treated) and control rats (DP-untreated).Non-diabetic control rats that did not develop diabetes had negligiblelevels of zonulin, consistent with the levels of zonulin shown in FIG.3. Moreover, two diabetic-prone rats that developed diabetes despitetreatment with the peptide antagonist of zonulin showed intraluminalzonulin levels that were significantly higher than the successfullytreated rats, and the untreated control rats. The levels of zonulin weresufficient to initiate the permeability changes necessary to progress todiabetes, but the ZOT/zonulin receptors were effectively blocked by thepeptide antagonist.

Also, following clinical presentation of diabetes, the intestinaltissues of the sacrificed rats were mounted in Ussing chamber to assessfor changes in ex vivo permeability.

More specifically, sections of jejunum and ileum were isolated from thesacrificed rats, and rinsed free of intestinal contents. Six sections ofeach intestinal segment was prepared and mounted in Lucite Ussingchambers (0.33 cm² opening), connected to a voltage clamp apparatus (EVC4000; World Precision Instruments, Saratosa, Fla.), and bathed withfreshly prepared buffer comprising 53 mM NaCl, 5.0 mM KCl, 30.5 mMNa₂SO₄, 30.5 mM mannitol, 1.69 mM Na₂PO₄, 0.3 mM NaHPO₄, 1.25 mM CaCl₂,1.1 mM MgCl₂, and 25 mN NaHCO₃ (pH 7.4). The bathing solution wasmaintained at 37° C. with water-jacketed reservoirs connected to aconstant temperature circulating pump and gassed with 95% O₂ and 5% CO₂.Potential difference was measured and short-circuit current and tissueresistance was calculated as described by Fasano et al, Proc. Natl.Acad. Sci. USA, 88:5242-5246 (1991). The results are shown in FIGS. 6-7.

As demonstrated in the ex vivo Ussing chamber permeability studies, andshown in FIG. 6, all of the rats that progressed to diabetes had anincrease in their intestinal permeability. Diabetic resistant (DR) ratshad no appreciable alterations in paracellular permeability (first bar).Untreated diabetic-prone rats (DP-untreated; second bar) had asignificant increase in paracellular permeability of the jejunum andileum. More importantly, diabetic-prone rats treated with the peptideantagonist of zonulin (DP-treated; third bar) had a significant increasein paracellular permeability of the small intestine restricted to thejejunum. However, as shown in FIG. 6, pre-treatment with the zonulinpeptide antagonist prevented these changes in the distal ileum.Consequently, alterations in paracellular permeability associated withthe pathogenesis are restricted to the ileum. Also, as shown in FIG. 6,there are no significant changes in permeability of the colon, whichcoincides, with the regional distribution of the zonulin receptordistribution.

These results were further validated by a comparison of ex vivointestinal permeability in the small intestines of untreateddiabetes-prone rats that either developed (DP-D) or did not developed(DP-N) diabetes (FIG. 7). While no significant changes in jejunal Rtwere observed between DP-D and DP-N rats, a significant lower Rt of theileal mucosa of DP-D rats was observed as compared to DP-N rats (FIG.7).

Thus, the following conclusions can be made: (1) the peptide antagonistwas able to effectively block the permeability changes required for thedevelopment of diabetes; and (2) in those rats treated with the peptideantagonist, the levels of intraluminal zonulin are 3-fold higher thanthe treated rats that did not develop diabetes. In this population oftreated rats that developed diabetes, the amount of peptide antagonistmay not have been enough to block a sufficient number of ZOT/zonulinreceptors necessary to prevent diabetes.

60% of the treated rats did not develop diabetes. In this population ofrats, the peptide antagonist of zonulin effectively prevented theincrease in intestinal permeability necessary for the onset of diabetes.As shown in FIG. 5, the treated rats had levels of intraluminal zonulincomparable with the untreated controls, but due to the presence of thepeptide antagonist of zonulin, the overall permeability the smallintestine was not altered enough to initiate the pathophysiologicchanges necessary for the progression to diabetes. Interestingly, asshown in FIG. 5, the one control animal that did not develop diabeteshad negligible levels of zonulin, further supporting the role of zonulinin the pathogenesis of diabetes.

Accordingly, an early event in the pathogenesis of diabetes in BB/Worrats involves changes in zonulin-mediated intestinal paracellularpermeability. Furthermore, inhibition of the zonulin signaling systemwith the use of peptide antagonists of zonulin prevents, or at leastdelays, the onset of diabetes.

While the invention has been described in detail, and with reference tospecific embodiments thereof, it will be apparent to one of ordinaryskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

1. A method for prevention or delay of onset of diabetes comprisingadministering to a subject in need of such prevention or delay of onset,a pharmaceutically effective amount of a peptide antagonist of zonulin,wherein said peptide antagonist binds to zonula occludens toxinreceptor, yet does not physiologically modulate the opening of mammaliantight junctions.
 2. The method of claim 1, wherein said peptideantagonist comprises amino acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, and SEQ ID NO:24.
 3. The method of claim 1, wherein said peptideantagonist is from 8-110 amino acids in size.
 4. The method of claim 3,wherein said peptide antagonist is from 8-40 amino acids in size.
 5. Themethod of claim 1, wherein said peptide antagonist comprises amino acidsequence SEQ ID NO:15.
 6. The method of claim 1, wherein said peptideantagonist consists of amino acid sequence SEQ ID NO:15.
 7. The methodof claim 1, wherein said diabetes is type I diabetes.
 8. The method ofclaim 1, wherein said peptide antagonist is administered as an oraldosage composition for intestinal delivery.